Journal oflmmunological Methods, 24 (1978) 75--78
75
© Elsevier/North-Holland Biomedical Press
USE OF LOGIT PAPER IN DETERMINATION OF SUPEROXIDE DISMUTASE ACTIVITY IN HUMAN BLOOD CELLS
YOHNOSUKE KOBAYASHI, SUSUMU OKAHATA, KYOJI TANABE and TOMOFUSA USUI
Department of Pediatrics, Hiroshima University School of Medicine, Hiroshima, Japan (Received 9 May 1978, accepted 15 May 1978)
Superoxide dismutase activity of the human blood cells was determined by the capacity to inhibit nitrite formation from hydroxylammonium chloride. Relationship between percentage of nitrite formation and amount of purified human erythrocyte superoxide dismutase was plotted on conventional graph paper, double log and logit papers. A comparison of the results showed that a linear relationship was obtained only with the logit paper, and thus a more accurate and simple estimation of the activity can be made on a single determination of the specimens.
INTRODUCTION
Superoxide dismutase (SOD), which catalyzes the dismutation of superoxide, O~, in the following reaction: 02 + O5 + 2H ÷ -~ H202 + 02, is ubiquitously present in cells which make use of molecular oxygen. In our series of experiments on SOD activity of blood cells, we determined such activity using either the m e t h o d of McCord and Fridovich (1969) or that o f Elstner and Heupel (1976). Difficulties were encountered when we attempted to obtain the a m o u n t of specimen required for 50% inhibition on either a conventional scale graph paper or on a c o m m o n l y used log--log paper. Thus, logit paper was employed to obtain the relationship of linearity between the extent of inhibition and the a m o u n t of purified erythrocyte SOD added to the reaction mixture. This m e t h o d of determination proved to be simple and easily performed. MATERIALS AND METHODS
Human blood was donated from healthy adult volunteers. L y m p h o c y t e s were separated by a differential centrifugation in a Conray-Ficoll solution, and granulocytes were obtained from the remaining pellet after sedimentation of erythrocytes by 3% dextran-saline solution. Contaminating erythrocytes were removed by hypotonic lysis. Purity of each cell preparation was more than 95%, respectively. Both granulocytes and lymphocytes were suspended in 0.1% Triton-X100 and disrupted by sonication for 5 min at
76
200 W (Insonator Model 200 M, Kubota, Japan). Sonicated materials were centrifuged at 25,000 X g for 30 min in the cold and clear supernatant was assayed for SOD activity. E r y t h r o c y t e specimen and purified erythrocyte SOD from humans were prepared by the m e t h o d described by McCord and Fridovich (1969). Protein c o n t e n t of the leukocyte and erythrocyte specimens was determined by the m e t h o d of Lowry et al. (1951) using bovine serum albumin as the standard. SOD activity was determined by the m e t h o d of Elstner and Heupel (1976), i.e., inhibition of nitrite formation from h y d r o x y l a m m o n i u m chloride. The a m o u n t of specimen required to inhibit the rate of nitrite formaLion by 50% was defined as 1 unit of activity (McCord and Fridovich, 1969). SOD activity was expressed as units per 1 mg of protein, respectively. R E S U L T S AND D I S C U S S I O N
Table 1 shows the percentage o f nitrite formation from hydroxyla m m o n i u m chloride with each indicated a m o u n t of purified human erythrocyte SOD. Figure 1A indicates the relationship between the a m o u n t of specimen and percentage of nitrite formation as plotted on conventional graph paper. Determination of the a m o u n t required for a 50% inhibition demands acquisition of values near 50%, actually within a range of +_5% at most, as the direct connection between the points outside this range did not allow for accurate estimation of the activity. When these same values are plotted on a log--log scale, the line tends to be more linear (Fig. 1B) than that in Fig. 1A but is not satisfactory from a quantitative point of view. When these values were plotted on logit paper (Fig. 1C, Rodbard et al., 1969), however, a linear relationship was obtained and the slope was the same for leukocyte and e r y t h r o c y t e specimens as well (data not shown). Determination of blood cell SOD was made in duplicate or triplicate.
TABLE 1 RELATIONSHIP BETWEEN NITRITE FORMATION AND AMOUNT OF PU RI FI E D H U M A N E R Y T H R O C Y T E SOD A m o u n t o f p u r i f i e d e r y t h r o c y t e SOD (units) 0
0.25
0.50
1.0
2.5
5.0
A 530n m
0.274 0.276 0.271
0.205 0.205 0.199
0.175 0.175 0.170
0.135 0.135 0.143
0.088 0.086 0.090
0.056 0.061 0.057
Mean
0.274
0.203
0.173
0.137
0.088
0.058
74
63
50
32
21
P e r c e n t a g e o f nitrite f o r m a t i o n
77
(B) LoG-LoG PAPER
100
70 60
50
50 40
30
20
2¢
oT,
,
0.251.0
,o
' 2.5
' 5.0
80%
(C)
10 0.25
' 0.5
' 1.0
i
2.5
, 5.0
LOGITPAPER
6O 5O 40 30 20
i i
T 0.25
, 0.5
, ,i
,
1.0 1.55 2 . 5
, 5.0
Fig. 1. R e l a t i o n s h i p o f n i t r i t e f o r m a t i o n a n d a m o u n t o f p u r i f i e d h u m a n e r y t h r o c y t e SOD o n (A) c o n v e n t i o n a l g r a p h p a p e r , (B) log--log p a p e r , a n d (C) logit paper. O r d i n a t e indicates p e r c e n t a g e n i t r i t e f o r m a t i o n a n d abscissa a m o u n t o f purified SOD in units.
Percentage of nitrite f or m at i on with a specimen was calculated and pl ot t ed on the ordinate and a corresponding SOD unit was obtained (Fig. 1C). An SOD activity o f the sample was expressed as units per mg o f protein. For example, when 0.2 ml o f a given specimen yielded 40% nitrite form at i on and contained 0.25 mg o f protein, the corresponding SOD unit was 1.55 units from the figure and c o n s e q u e n t l y SOD unit o f the sample is 1.55/0.25 = 6.2 units/mg o f protein. T w o ot her different volumes of the same specimen, c o n s e q u e n t l y with different amounts o f protein as the d e n o m i n a t o r , p r o d u c e d m u c h the same results. In this way, a single determination enables acquisition o f the correct a m o u n t of SOD units with a relatively small a m o u n t o f specimen. Normal values o f SOD activities o f e r y t h r o c y t e s and
TABLE 2 NORMAL VALUES OF ERYTHROCYTE HUMAN BLOOD
units/rag protein
A N D L E U K O C Y T E SOD A C T I V I T I E S O F
E r y t h r o c y t e (10) a
G r a n u l o c y t e (10)
Lymphocyte (10)
6 . 8 7 5 +- 1 . 4 0 0 b
6.4 -+ 1.4
13.7 -+ 4.0
a N u m b e r in p a r e n t h e s e s i n d i c a t e s t h e n u m b e r of s u b j e c t s s t u d i e d . b M e a n +_ 1 SD.
78 leukocytes from healthy adults are shown in Table 2. Such are almost 2.5 times higher in activity than those determined by the c y t o c h r o m e c reduction inhibition m e t h o d . ACKNOWLEDGMENT This study was supported in part by a research grant of 'Immunodeficiency disorders' and 'Angitis' from the Ministry of Health and Welfare and Research Grants (Project No. 157253 and 144052) from the Ministry of Education, Science and Culture, Japan. Miss M. Ohara, K y o t o University, assisted in the preparation of the manuscript. REFERENCES Elstner, E.F. and A. Heupel, 1976, Anal. Biochem. 70,616. Lowry, O.H., N.J. Rosebrough, A.L. Farr and R.J. Randall, 1951, J. Biol. Chem. 193, 265. McCord, J.M. and I. Fridovich, 1969, J. Biol. Chem. 244, 6049. Rodbard, D., W. Bridson and P.L. Rayford, 1969, J. Lab. Clin. Med. 74,770.
75
© Elsevier/North-Holland Biomedical Press
USE OF LOGIT PAPER IN DETERMINATION OF SUPEROXIDE DISMUTASE ACTIVITY IN HUMAN BLOOD CELLS
YOHNOSUKE KOBAYASHI, SUSUMU OKAHATA, KYOJI TANABE and TOMOFUSA USUI
Department of Pediatrics, Hiroshima University School of Medicine, Hiroshima, Japan (Received 9 May 1978, accepted 15 May 1978)
Superoxide dismutase activity of the human blood cells was determined by the capacity to inhibit nitrite formation from hydroxylammonium chloride. Relationship between percentage of nitrite formation and amount of purified human erythrocyte superoxide dismutase was plotted on conventional graph paper, double log and logit papers. A comparison of the results showed that a linear relationship was obtained only with the logit paper, and thus a more accurate and simple estimation of the activity can be made on a single determination of the specimens.
INTRODUCTION
Superoxide dismutase (SOD), which catalyzes the dismutation of superoxide, O~, in the following reaction: 02 + O5 + 2H ÷ -~ H202 + 02, is ubiquitously present in cells which make use of molecular oxygen. In our series of experiments on SOD activity of blood cells, we determined such activity using either the m e t h o d of McCord and Fridovich (1969) or that o f Elstner and Heupel (1976). Difficulties were encountered when we attempted to obtain the a m o u n t of specimen required for 50% inhibition on either a conventional scale graph paper or on a c o m m o n l y used log--log paper. Thus, logit paper was employed to obtain the relationship of linearity between the extent of inhibition and the a m o u n t of purified erythrocyte SOD added to the reaction mixture. This m e t h o d of determination proved to be simple and easily performed. MATERIALS AND METHODS
Human blood was donated from healthy adult volunteers. L y m p h o c y t e s were separated by a differential centrifugation in a Conray-Ficoll solution, and granulocytes were obtained from the remaining pellet after sedimentation of erythrocytes by 3% dextran-saline solution. Contaminating erythrocytes were removed by hypotonic lysis. Purity of each cell preparation was more than 95%, respectively. Both granulocytes and lymphocytes were suspended in 0.1% Triton-X100 and disrupted by sonication for 5 min at
76
200 W (Insonator Model 200 M, Kubota, Japan). Sonicated materials were centrifuged at 25,000 X g for 30 min in the cold and clear supernatant was assayed for SOD activity. E r y t h r o c y t e specimen and purified erythrocyte SOD from humans were prepared by the m e t h o d described by McCord and Fridovich (1969). Protein c o n t e n t of the leukocyte and erythrocyte specimens was determined by the m e t h o d of Lowry et al. (1951) using bovine serum albumin as the standard. SOD activity was determined by the m e t h o d of Elstner and Heupel (1976), i.e., inhibition of nitrite formation from h y d r o x y l a m m o n i u m chloride. The a m o u n t of specimen required to inhibit the rate of nitrite formaLion by 50% was defined as 1 unit of activity (McCord and Fridovich, 1969). SOD activity was expressed as units per 1 mg of protein, respectively. R E S U L T S AND D I S C U S S I O N
Table 1 shows the percentage o f nitrite formation from hydroxyla m m o n i u m chloride with each indicated a m o u n t of purified human erythrocyte SOD. Figure 1A indicates the relationship between the a m o u n t of specimen and percentage of nitrite formation as plotted on conventional graph paper. Determination of the a m o u n t required for a 50% inhibition demands acquisition of values near 50%, actually within a range of +_5% at most, as the direct connection between the points outside this range did not allow for accurate estimation of the activity. When these same values are plotted on a log--log scale, the line tends to be more linear (Fig. 1B) than that in Fig. 1A but is not satisfactory from a quantitative point of view. When these values were plotted on logit paper (Fig. 1C, Rodbard et al., 1969), however, a linear relationship was obtained and the slope was the same for leukocyte and e r y t h r o c y t e specimens as well (data not shown). Determination of blood cell SOD was made in duplicate or triplicate.
TABLE 1 RELATIONSHIP BETWEEN NITRITE FORMATION AND AMOUNT OF PU RI FI E D H U M A N E R Y T H R O C Y T E SOD A m o u n t o f p u r i f i e d e r y t h r o c y t e SOD (units) 0
0.25
0.50
1.0
2.5
5.0
A 530n m
0.274 0.276 0.271
0.205 0.205 0.199
0.175 0.175 0.170
0.135 0.135 0.143
0.088 0.086 0.090
0.056 0.061 0.057
Mean
0.274
0.203
0.173
0.137
0.088
0.058
74
63
50
32
21
P e r c e n t a g e o f nitrite f o r m a t i o n
77
(B) LoG-LoG PAPER
100
70 60
50
50 40
30
20
2¢
oT,
,
0.251.0
,o
' 2.5
' 5.0
80%
(C)
10 0.25
' 0.5
' 1.0
i
2.5
, 5.0
LOGITPAPER
6O 5O 40 30 20
i i
T 0.25
, 0.5
, ,i
,
1.0 1.55 2 . 5
, 5.0
Fig. 1. R e l a t i o n s h i p o f n i t r i t e f o r m a t i o n a n d a m o u n t o f p u r i f i e d h u m a n e r y t h r o c y t e SOD o n (A) c o n v e n t i o n a l g r a p h p a p e r , (B) log--log p a p e r , a n d (C) logit paper. O r d i n a t e indicates p e r c e n t a g e n i t r i t e f o r m a t i o n a n d abscissa a m o u n t o f purified SOD in units.
Percentage of nitrite f or m at i on with a specimen was calculated and pl ot t ed on the ordinate and a corresponding SOD unit was obtained (Fig. 1C). An SOD activity o f the sample was expressed as units per mg o f protein. For example, when 0.2 ml o f a given specimen yielded 40% nitrite form at i on and contained 0.25 mg o f protein, the corresponding SOD unit was 1.55 units from the figure and c o n s e q u e n t l y SOD unit o f the sample is 1.55/0.25 = 6.2 units/mg o f protein. T w o ot her different volumes of the same specimen, c o n s e q u e n t l y with different amounts o f protein as the d e n o m i n a t o r , p r o d u c e d m u c h the same results. In this way, a single determination enables acquisition o f the correct a m o u n t of SOD units with a relatively small a m o u n t o f specimen. Normal values o f SOD activities o f e r y t h r o c y t e s and
TABLE 2 NORMAL VALUES OF ERYTHROCYTE HUMAN BLOOD
units/rag protein
A N D L E U K O C Y T E SOD A C T I V I T I E S O F
E r y t h r o c y t e (10) a
G r a n u l o c y t e (10)
Lymphocyte (10)
6 . 8 7 5 +- 1 . 4 0 0 b
6.4 -+ 1.4
13.7 -+ 4.0
a N u m b e r in p a r e n t h e s e s i n d i c a t e s t h e n u m b e r of s u b j e c t s s t u d i e d . b M e a n +_ 1 SD.
78 leukocytes from healthy adults are shown in Table 2. Such are almost 2.5 times higher in activity than those determined by the c y t o c h r o m e c reduction inhibition m e t h o d . ACKNOWLEDGMENT This study was supported in part by a research grant of 'Immunodeficiency disorders' and 'Angitis' from the Ministry of Health and Welfare and Research Grants (Project No. 157253 and 144052) from the Ministry of Education, Science and Culture, Japan. Miss M. Ohara, K y o t o University, assisted in the preparation of the manuscript. REFERENCES Elstner, E.F. and A. Heupel, 1976, Anal. Biochem. 70,616. Lowry, O.H., N.J. Rosebrough, A.L. Farr and R.J. Randall, 1951, J. Biol. Chem. 193, 265. McCord, J.M. and I. Fridovich, 1969, J. Biol. Chem. 244, 6049. Rodbard, D., W. Bridson and P.L. Rayford, 1969, J. Lab. Clin. Med. 74,770.
